Novel process for producing antibiotics bleomycin

ABSTRACT

GROUP AND AT LEAST ONE BASIC GROUP SELECTED FROM AMINO, IMINO, GUANIDINO, AMIDINO AND SULFONIUM GROUPS AND NITROGEN-CONTAINING CYCLIC COMPOUNDS, OR IN THE PRESENCE OF A COMPOUND CONVERTIBLE IN THE CULTURE LIQUOR TO SUCH AMINO COMPOUND AS MENTIONED ABOVE, THEREBY SELECTIVELY PRODUCING A KNOWN OR NOVEL BLEOMYCIN COMPONENT CORRESPONDING TO THE SAID AMINO COMPOUND OR TO AN AMINO COMPOUND DERIVED FROM THE ABOVE-MENTIONED COMPOUND, AND THEN THE KNOWN OR NOVEL BLEOMYCIN IS RECOVERED BY KNOWN MEANS FROM THE CULTURE MEDIUM.   A novel process for producing antibiotics bleomycins by innoculating and aerobically culturing in a nutrient sourcecontaining medium a bleomycin-producing strain belonging to the actinomyces, wherein the culture is effected in the presence of an amino compound having at least one

United States Patent 1191 Umezawa et al.

[451 Nov. 5, 1974 NOVEL PROCESS FOR PRODUCING ANTIBIOTICS BLEOMYCIN [75]Inventors: Hamao Umezawa; Kenii Maeda,

both of Tokyo; Tomohisa Takita, Asaka; Yuya Nakayama, Yono; Akio Fujii,Tokyo; Nobuyoshi Shimada, Tokyo; Hideo chimura, Tokyo, all of Japan [73]Assignee: Zaidan Hojin Biseibutsu Kagaku Kenyu Kai, Tokyo, Japan [22]Filed: Sept. 21, 1972 [21] Appl. No.: 291,079

Related US. Application Data [63] Continuation-in-part of Ser. No.10,421, Feb. 11,

1970, abandoned.

[52] US. Cl 260/ll2.5, 195/80 R, 260/239 A, 260/247.2 A, 260/256.4 R,260/268 H,

H, 260/309, 260/326.8, 260/345.l, 424/177, 260/210 AB [51] Int. Cl...C07c 103/52, A61k 27/00, C0721 7/00 [58] Field 01' Search 260/1 12.5

[56] References Cited FOREIGN PATENTS OR APPLICATIONS OTHER PUBLICATIONSUmezawa, Asian Med. 1., 13, 190-209, (1970). Takita et al., Prog. inAntimicro.

Germany Anticancer Chemother., 2, 1031-1036, (1970). Takita et al., J.of Antibiotics (Tokyo), 22, 237239, (1969).

Primary ExaminerLewis Gotts Assistant Examiner-Reginald J, SuyatAttorney, Agent, or FirmSchuyler, Birch. Swindler, McKie & Beckett [57]ABSTRACT A novel process for producing antibiotics bleomycins byinnoculating and aerobically culturing in a nutrient source-containingmedium a bleomycin-producing strain belonging to the actinomyces,wherein the culture is effected in the presence of an amino compoundhaving at least one 1 Claim, 6 Drawing Figures PATENTED NOV 5 197:

SHEET 30F 6 @Sm 8Q DMGN QUQW

sla4sl4oo SHEET 5 OF 6 NOVEL PROCESS FOR PRODUCING ANTIBIOTICS BLEOMYCINCROSS REFERENCE TO THE RELATED APPLICATION This application is acontinuation-in-part application of US. Ser. No. 10,421 filed on Feb.11, 1970, now abandoned.

This invention relates to a novel process for producing antibioticsbleomycins. More particularly, the invention pertains to a novel processfor producing antibiotics bleomycins by inoculating and aerobicallyculturing in a nutrient source-containing medium a bleomycin-producingstrain belonging to the actinomyces, wherein the culture is effected inthe presence of an amino compound having at least one per sulfate, etc.,and the resulting bleomycin is isolated and recovered in the form ofhydrochloride or sulfate from the culture liquor by adoption of such ameans as using ion-exchange resin. They contain A A A' A A A A B B B B BB and the like components (hereinafter referred to as the bleomycincomponents) and, when further subjected to copper removal treatment,these bleomycin components are obtained as white powders. Bleomycin hasbeen known as a general term for antibiotics containing these components(British Pat. No. 1,038,242).

When the culture is effected under ordinary conditions, these bleomycincomponents are produced simultaneously, but the ratio of individualcomponents is not definite and belomycins A and B are chiefly producedin most cases.

With an aim to attain a process in which desired bleomycin componentsamong those mentioned above can be selectively produced with favorableyields, the present inventors studied the structures of bleomycins to 33H NH find that the bleomycin components have a structure re resented bythe formula (I) set forth below, P e

O NH

2 ll lH NH H C N-CH c 2 2 O O N N f a if 3 ,0 HO ,c1-1 ,N ,0 I N R c 0on e NH H NH g N S 2 CH l l CH 0 cn on A H CH N CH 2 H H 3 3 2 CH H H I1H CH O 2 H W O 1 (p H- H 0 C on H H cn on C CpC OH H/OH 2: I l

0 NH group and at least one basic group selected from wherein Rrepresents any of the formulas amino, imino, guanidino, amidino andsulfonium 1 groups and nitrogencontaining cyclic compounds, or in l CH R,the presence of a compound convertible in the culture 2 2 liquor tosuch amino compound as mentioned above, thereby selectively producing aknown or novel bleomycin component corresponding to the above- (9 1+mentioned amino compound or to an amino compound (2 i derived from theabove-mentioned compound, and 5 i R then the known or novel bleomycin isrecovered by known means from the culture medium. 3 Bleomycins areanti-tumor antibiotics discovered by 9 Umezawa et al. and are obtainedin such a manner that 3 -NH-CH -X- R a bleomycin-producing strainbelonging to the actinomyces Streptomyces verticillus (e.g., ATCC No.15003) 4) NH CH X N d is aerobically cultured with stirring in anutrient me 2' an ium ihlQYE EJ,REEEE LPEQQBDQW: iI.. EL EEE ,P: ose),glucose, soybean power, corn steep liquor, so- (5) -Nl-I-CH -X' 1 I diumchloride, potassium phosphate, zinc sulfate, copwher e X, XTR, R R R andR are as hereinafter defined, and that R in the formula varies, forexample below, depending on the kind of bleomycin components.

I NH

Based on the above-mentioned knowledge, the inventors further repeatedthe studies to find that when various amino compounds corresponding tothe side chain portion R in the above-mentioned formula (I), or aminocompounds convertible during the culture to amines corresponding to theside chain portion, are added to the culture liquors ifbleomycin-producing strains, desired bleomycin compounds can be obtainedin favorable yields, and that when certain kinds of amino compounds areadded, novel bleomycins differ ent from the aforesaid bleomycins areproduced and, when separated from the culture liquors and are purified,the novel bleomycins can be recovered in high yields. On the basis ofthe above findings, the present inventors have accomplished the presentinvention.

The aforesaid amino compounds used in the present invention may berepresented by the following five general formulas:

wherein X -(CH -NH-(CH -NH-(CH --NH wherein Y n, n n n, n', 71"", n'" aninteger of 1-8,

n an integer of 0-8,

Rl0;Rl0" R16 The dotted line in the formula (for example etc.) showsthat hydrogen atom on carbon chain may 1, 2 D1aminoethane l,B-Diaminopropa'ne l, l-Diaminobutane l, 5-Diaminopentane l,6-Diaminohexane N- 2 Aminoethyl )l, diaminoethane N- 3-Aminopr'opyl )-l,3- diaminopr-opane N- 3-Aminopr'opyl )-l, diam ino butane (spermidine)N- 3-Aminopr-opyl )-l 6- diaminohexane N-(5-Aminopentyl )-1, 5-diaminopentane N, N -Bis 2-amino ethyl di-aminomethane N, N'-B1s(2-am1noethy1 l, 2-diaminoethane N, N' -Bis(3-aminopropyl 1,l-diaminobutane (spermine) N, N -Bis(3-aminopr'opyl 1,6-d1am1nohexane N,N' -B1s( 5-aminopentyl l, 5-diaminopentane 2, 2, B-Trimethylpentamethylenediamine l, Q-Diaminopropane N-Methyl-l, 3-diaminopropane N-Butyl-l,B-diaminopropane N-Ootyl-l, 3-diaminopropane N, N-Dimethyl-l, 2- d1amino ethane CH CH 13 14. N, N-Dirne'chyl-N' -propionyl- ./CH 1,3-diaminopropane CQHB'CO'NH' CH2 )3'N\ CH 3 /CHl-Carboxyl-dimethylamino- NHQ-CH- CH2) -N 3 butylamine 3 CH coon 3 /CH3l-Carbamoyl-4-dimethy1amino- NH2-CH-(CH2 3-N\ butylamine I CH CONH2 3 N,N-Dimethyl-N -benzoyl- 1 3-diaminopropane propyl )-l, 3-diaminopr'opaneWhen 3-aminopropyl-dimethylsulfonium bromide or 73-amino-3-carboxypropyl-dimethylsulfonium chloride, for example, isadded as the amino compound to a nutrient medium and suchbleomycin-producing strain as Streptomyces verticillus is cultured'insaid medium, the productivity of bleomycin A is enhanced to increase thecontent (percent) of bleomycin A among the whole bleomycins present inthe culture liquor. Likewise, the addition of hydrochloride of agmatineincreases the content (percent) of bleomycin B the addition of 1,4-diaminobutane increases the content (percent) of bleomycin A and theaddition of spermidine increases the content of bleomycin A However, theaddition of spermine results not only in the production of bleomycin Aof corresponding side chain but also in the production of bleomycin Ahaving in the side chain spermidine which has been formed from thespermine during the culture. In the case of a strain having strongability of converting spermine to spermidine, the addition of sperrnineresults chiefly in the production of bleomycin A Likewise, the additionof, for example, 1,2-diaminoethane or 1,3-diaminopropane results in theproduction of novel Z-aminoethylaminobleomycin or3-aminopropylamino-bleomycin. Further, such a chain polyamino compoundas N,N-bis(3- aminopropyl)-methylamine chiefly produces such novelbleomycin as 3-(N-methyl-N-3-aminopropyl)- aminopropylamino-bleomycinhaving the same side chain as that of the compound added and, inaddition, entrains a low molecular weight amine, which has been formedfrom said additive during the culture to byproduce3-N-methylaminopropylamino-bleomycin.

That microorganisms have such abilities as ester decomposition,dealkylation and decarboxylation has been well known, and aminocompounds which convert during the culture to the amines of bleomycinside chains are involved in the present invention like the aminocompounds represented by the aforesaid general formulas (l) -(5). Forexample, compounds, in which NH in the aforesaid general formulas(l)-(5) has been acylated or alkylated, or hydrogen of the methylenegroup in the 2-position of NH has been substituted by a carboxylic groupor an acid amide group and which are easily metabolized to amines, canbe used like the amino compounds represented by the aforesaid generalformulas (l )-(5 Generally, the amino compounds to be added are usedeither as they are or in the form of inorganic acid salts, but theconcentrations thereof in the culture liquor vary depending on theirkind. In general, the concentration of the amino compounds is within therange of 01-45 milligram per milliliter of the culture liquor.

The process of the present invention is carried out in the followingmanner:

In practicing the present process, it is preferable to use a mediumwhich is composed mainly of carbohydratesand nitrogen-containing organicsubstances such as millet jelly, glucose, starch, soybean powder, cornsteep liquor, etc., and which has been incorporated with small amountsof inorganic substances such as, for example, potassium phosphate,copper sulfate, zinc sulfate, sodium chloride, nitrates, etc. Amongabovementioned substances, the carbohydrates and the nitrogen-containingorganic substances are used in an amount of 01-10 percent by weight andthe inorganic salts are used in an amount of 0.01-5 percent by weight,based on the total Weight of the medium. In this me dium, Str eptomyc es ve rticillus or a bleomycinproducing strain among the variantsthereof, such as for examples, the strains ATCC 15003 or ATCC 21678, isaerobically cultured at 25-30C and pH of 5.0-9.0 for 50-300 hoursaccording to ordinary procedures, which have been shown in theliterature, l-l. Umezawa et al., New Antibiotics Bleomycin A and B, J.Antibiotics, Ser. A, 1966, pp. 200-209. Thereafter, the culture liquoris subjected to treatment with ordinary procedures, which have beenshown in the literature, H. Umezawa et al., Purification of Bleomycins,J. Antibiotics, Ser. A, 1966, pp. 210-215, to recover bleomycinsproduced. That is, the culture liquor is filtered and the filtrate isadsorbedon, for example, a cation exchange resin having a reactivegroup, such as a carboxyl group, and is then eluted with an aqueoushydrochloric acid solution. The cation exchange resin includes, such asfor example, Amberite lRC-SO and Duolite CS-lOl (tradename for acidiccation exchange resin containing carboxylic acid group, the former ismanufactured by Rohm & Haas Co., U.S.A., the latter by Chemical ProcessCo., U.S.A.). Subsequently, the elute is subjected successively to thesteps of adsorption on active carbon, elution with aqueousacetoneufactured by Pharmacia Fine Chemicals Inc.) as a carrier.Finally, the eluate is adsorbed on active carbon and is subjected to thesteps of water-washing and elution with aqueous acetone-hydrochloricacid solution,

hydrochloric acid solution, alumina adsorption, aquewhereby a purebleomycin component can be obtained. ous methanol elution, adsorption onSephadex (tradename for a dry, insoluble powder composed of micro- If,in accordance with the present invention, a bleoscopic beads which aresynthetic, organic compounds mycin component has sufficientlyselectively been proderived from the polysaccharide degtt rani ma faduced in the culture medium, the step of separation tured and sold byPharmacia Fine Chemicals Inc.) and using CM Sephadex may be omitted.elution with distilled water, whereby a bleomycin hy- Bleomycins have aproperty to chelate with copper drochloride powder is obtained. Forfurther purificaand are obtained as a blue powder from the culturelition of the thus obtained powder, it is preferable to quor, but thecopper can be removed by effecting the adopt a means of chromatographicelution with ammotreatment of copper removal in any of the extraction ornium chloride or aqueous ammonium formate solution, purification step.using CM Sephadex (tradename for an acidic ion- Several properties ofbleomycins obtained according exchanger composed ofcarboxymethyl-Sephadex manto the present invention are set forth inTable 1.

TABLE 1 (m ir a lfi n i ggnt ifi'the time" bleomycins obtained by NumberName and physical property of additive Name and side chain structure ofnew (main) bleomycin 1 B-aminopropyl-dimethylsulfonium bromidehydrobromide, 72 Bleomyein A2.

M.P. 8788 C.

2 S-amino-3-carboxypropyldimethylsulfonium chloride, 87 Do.

M.P. 130-140 C. (decomp.).

3 Agmatine sulfate, M.P. 231 C 76 Bleomycin B2.

4 1,2-diaminoethane, B.P. 116-117 C 80 2-amin0ethylamino-bleomycin, NH(Cl-I2)2NII.

5 1,3-diaminopropane, B.P. 135136 0-0.. t. 3-aminopropylamino-bleomycin,NH2(CH2)aNH- 6 lA-diaminobutane, B.P. 158160 C 85 Bleomyein A'g.

7.. Spermidine, B.P. 115 (1/3 mm. Hg Bleomyein A5.

8 Spermine, B.P. 150-160 C./3 mm. Hg 91 Do.

U N ,N-dimethy1-1,2-diaminoethane, B.P. 108 C J13-N,N-dimethylaminoethylarniuobleon1yein N(CHz)2NH 1UN,N-diethyl-l,2-diaminoethane, B.P. 147151 C t. 843-N,N-diethylaminoethylamino-bleomycin N (OH2)2NH- a W MW s, s ,7 A, H5

1] N,N diethy1-1,3-diamin0propane, B.P. 1fi6172 C 4. 953-N,N-diethylarninopropylamino-bleomycin N(CH2);NH-

Cal-I5 12 N-butyl-N-(3-aminopropyl)-1,3-diaminopropane, B.P. 963-(3-N-butyl-aminopr0pyl)-aminopropylamino-bleomycin,

-126 C./3 mm. Hg. C4H9NH CIIZ 3NH(C11I2)3NH.

13 N-C(2;}i2y;dIfI gryI1{)rgopyl)-1,Q-diaminoethane, B.P. 123126 952-(2-hydroxypropyl)-aminoethylamino-bleomycin CIIZCJTICHZN}I(CI{Q)ZNI{14 N-(S-aminopropyl)-piperazilie, B.P. 8081 C./2 mm. Hg. 803-(piperazino)propylamino-bleomycin lIN RY-(o1h)aNIll5 N-(l-plu nyltthyl)-N-(3-un\inopropyU-l,3-(liumino- 72 3l3-(N-l-plmnyl t.hl)-mninopropylhuninopropylumin0- propmm, B.P. HIP-153 5 mm. .llg.llltOlllyClll 0 (1llNll((7lIg)aNl[(t).lI2)aNIl llln 16, N,1,2-diaminopropune, B.P. 118-11J C J0 Z-aminopropylamino-bleomycinProperties of new (main) bleomycins hydrochloride Elementary analysis(percent) Chromatography(R1) Reaction Ultmviqlel. Potency absorptmnNumber C H N S (mcg./mg.) Thin layer Paper N S Appemmlcv111:1Xi1111l111(1llp) 1 4 1 0.30 15. 0 4. 00 1:888 0.40 0.87 W00 8, 1243.00 0.02 10.40 3 781 0.23 0.84 13 43.50 0.45 10.38 4.351 0.05 0.84 1444.01 0.21 10.50 3 01{ 0.30 0.84 ..00 15 45.51 0.42 10.45 3.20{ 0.400.75 10 43.80 0.04 10.00 4 31 1 0.00 0.81 17 43.07 0.24 10.42 418{ 888}0.51 0.83 .....110 18 43.30 0.31 10.20 4.32 8:188 0. 70 0.83 10 10 44.100.35 10.30 4.00{ 0.28 0.83 10 20 44. 71 0. 41 10.21 4 0c 1 888 1 0.38 0.80 10 21 43,80 0.35 10.01 4.05 8:888} 0.14 0.83 .do 22 43. 20 0.20 17.004 00 1 0.23 0. 83 .d0 23 43.42 0.25 10.80 4 11 1 0.32 0.84 10 24 44.000.22 10.20 4 12 1 '888 0.30 0.88 ..do g8: 25 44.71 0.38 10.17 4.21 '88;1 0.14 0.80 ..do 32: 26 4 .30 0. 25 10.15 4 03 1 1 0.05 0. 83 27 44.380. 10.78 3.05 888 0.30 0.85 .....do 381 28 44.01 0.14 17.52 3 00 1;: ,880.25 0.80 do .38: 20 44.20 0.41 10.80 3.08 1 8:88; 1 0.1 0.88 .-d0 3044.30 0.51 10.02 1.00 81288 1 0.14 0. 88 --.-.d0 1:321 31 44.52 0.30 10.20 3.80 1 0.38 0.87 do 32 43,70 0.40 10.38 4.221 8:888} 0.03 0.88 010 3843.01 0.35 10.21 4.10 0. 34 0.87 34 45.40 0. 41 15.30 4.00 888 0.53 0.7032,1 35 45.30 0.42 15.13 4.13 1 0.02 0.85 30 44.80 0.28 15.08 4.10 0.010.00 321 37 44.80 0.30 15.70 4.18 8:888 0.58 0.04 321 38 44.01 0.0015.27 4.031 0.57 0.70 00 30 44. 02 0.03 10.48 4.02 88:88? 0.24 0.80.'...do 40 40.30 0.37 16.48 4.35 1 1 0.07 0.83 .d0 41 44. 00 0.43 10. 204.25 1 0. 41 0.72 .....d0 42 43.85 0.25 10.80 4.281 0.40 5 0.70 do 4343.27 0.50 17.40 4.22 1 0.33 0.70 882- 44.-.. 43.00 0.34 10.32 4.20 10.04 0.81 .00... 48.20 0. 28 10.50 4.28 1 0.57 0. ..410..- 40 44.500.30' 15.81 4.08 1 1 0.53 0.88 10 47 44.30 0.40 15.00 4.03 1 0. 00 0.7000 48 44.40 0.50 15.72 4.311 0.25 0.80 d0 TABLE 1C0ntin u d I M Infra,red absorption, wave number (cm.-

TABLE l Continued N mber Infra red absorption, wave number (cmr 3, 330.2, 920 1, 716 1, 641 1, 560 1,462 1,370 1,005 1,053 1, 000 3, 331 2,021 1, 715 1, 640 1, 558 1, 460 1, 370 1, 005 1, 055 1, 000 3,320 2, 9101, 715 1,640 1,551 1,460 1,365 1, 005 1,050 1,005 3,310 2, 020 1,7101,647 1, 557 1,456 1,371 1,003 1,051 1,007 3, 332 2, 030 1, 720 1, 6401, 555 l, 460 1, 370 1,005 1, 050 1, 005 3, 340 2, 045 1, 720 1, 642 1,560 1, 462 1,370 1, 0515 1, 050 1, 005 3,310 2, 040 1, 721 1,641 1,5561,458 1,370 1, 005 1,057 1,010 3, 348 2,020 1, 716 1, 647 1, 555 1, 4601, 372 1, 000 1, 053 1,008 3, 340 2, 020 1, 721 1, 646 1, 560 1, 457 1,375 1, 005 1, 053 1, 006 3, 372 2, 026 1, 710 1, 642 1, 554 1, 465 1,370 1, 000 1, 050 1, 007 3,380 2,022 1,715 1, 64'.) 1,560 1,466 1,373 1,002 1,052 1,010 3,375 2,930 1,716 1,650 1,562 1,470 1,375 1,100 1,0531,007 3, 300 2, 020 1, 722 1, 662 1, 555 1, 467 1, 372 1, 092 1,0501,008 3, 321 2, 027 1, 710 1, 660 1, 557 1, 462 1,370 1, 005 1,052 1,010 3, 350 2, 040 1, 721 1, 648 1, 557 1, 460 1, 372 1, 005 1,052 1,0083, 350 2, 010 1, 718 1, 645 1, 560 1, 463 1, 375 1,001 1,055 1, 010 3,341 2, 021 1, 725 1, an 1,558 1, 455 1, 375 1, 093 1, 057 1,008 3,3402,010 1,721 1,642 1,555 1,457 1,372 1, 005 1,052 1,006 3, 346 2, 920 1,718 1, 640 1,557 1, 450 1,370 1, 005 1,050 1,005 3,300 2,950 1,721 1,6501,551 1,458 1,370 1,008 1,050 1,000 3, 320 2, 052 1, 720 1, 648 1,550 1,452 1,370 1, 095 1,051 1, 010 3, 340 2, 040 1, 710 1, 642 1, 550 1, 4501, 367 1, 000 1,040 1,005 3, 350 2, 047 1, 720 1,645 1, 552 1, 451 1,368 1, 090 1,052 1, 010 3,340 2, 050 1,720 1,640 1,551 1,456 1,373 1,0041,050 1,008 3, 320 2, 010 1, 725 1, 650 1, 550 1, 465 1,370 1, 005 1,050 1, 000

The infrared absorption spectrum and ultraviolet absorption spectrum ofseveral compounds in Table l are shown as examples in the attached FIGS.1-6 as follows:

1. The content of new (or main) bleomycin was measured in-such a mannerthat the bleomycin (mixture of individual components) obtained by theculture and purification in each of the examples shown previously wasseparated by use of CM-Sephadex and was then subjected to ultravioletabsorption spectrophotometry.

2. The potency was measured according to biological assay, usingcopper-free bleomycin A hydrochloride (potency 940 meg/mg) as a standardsubstance and Mycabacterium tuberculosis 607 (upper) and Bucillussubtilis (lower) as a test microorganism.

3. Thin layer chromatography:

Absorbing agent silica gel Solvent :9:1 mixture of methanol 10 percentammonium acetate-10 percent aqueous ammonia solution. Paperchromatography:

Solvent 10 percent aqueous ammonium chloride solution. 4. N ninhydrinreaction, S Sakaguchi reaction Novel bleomycins obtained according tothe present invention show no definite melting point, decompositionpoint or boiling point and inhibit gram positive and negative bacteriaand have antitumor activity.

Said bleomycins are soluble in water and aqueous methanol.

Said bleomycins form salts with acids and chelate with copper.

Said bleomycins give positive reaction in Pauly, Ehrlich, Dragendorffand permanganate reactions but give negative reaction in Molish, Biuret,Elson-Morgan, Maltol, Fehling, Tollens, Anthrone and ferric chloridereactions.

Said bleomycins exhibit, in infrared region, adsorption zone at thefollowing wave numbers: 1,0401,075 cm", 1,7l0-1,735 cm, l,625-l,680 cm,2,9l0-2,940 cm", 3,2003,400 cm.

Said bleomycins are further characterized by the facts that their acidhydrolysis give L-threonine,B-amino-B-(4-amino-6-carboxy-S-methyl-pyrimidine-Z-yl)- propionic acid,4-amino-3-hydroxy-Z-methyl-n-valeric acid, B-hydroxyhistidine,L-B-aminoalanine, L-glucose, 3-o-carbamoyl-D-mannose,2-(2-aminoethyl)-2,4'- bithiazole-4-carboxylic acid and each aminocompound corresponding to side chain portion.

As is clear from Table l and above-mentioned facts, the novel bleomycinsobtained according to the present invention are similar to the knownbleomycin components in appearance and infrared and ultravioletabsorption curve, but are obviously different therefrom in potency(mcg/mg) and Rf values. Further, what is worthy of special mention isthat when these novel substances are hydrolyzed with strong acid and thehydrolyzates are subjected to gas chromatography, the amino compoundsused as additives for the culture, or amino compounds formed during theculture, are detected and it has been confirmed that they have sidechains corresponding to the amino compounds added or to the aminocompounds formed during the culture.

In accordance with the present invention, it has first become possiblethat among the bleomycin components, those which are suitable forindividual therapeutic purposes can selectively be recovered in highyields and in pure forms. For example, bleomycin A which is strong inanti-tumor property and less in toxicity, can selectively obtained.Further, it has become possible to obtain such novel bleomycins as 3-(l-phenylethyl)- aminopropylamino-bleomycin which are excellent in effectand less toxicity.

In order to shown the fact that bleomycins obtained according to thepresent invention have a more safety and excellent anti-tumor effect ascompared with any other conventionally known anti-tumor substances, the

life-prolongation effect of bleomycins obtained according to the presentinvention is compared with that of mytomycin C as a control medicine onmice inoculated with the ascites type of the Ehrlich carcinoma below.

Method of experiment:

Mice of ICR/JCL strain (2 weeks old, five for one group) are inoculatedwith Ehrlich ascites carcinoma cells in a dose of 2 millions/mouse.

Bleomycin ranging from 25 mg/kg to 0.19 mg/kg is administeredintraperitoneally once a day for days starting from 2 hours after theinoculation, the death is observed everyday up to 50 days after theinoculation and body weights are measured every 5 days.

Judgement of the effect:

LD is calculated by Behres-Karbers method from the group poisoned athigh dose levels. A survival percentage at each dose level is obtainedby assuming the average number of survival days observed with thecontrol group administered with physiological saline water to be 100percent, and those having survived for such periods as 200 percent ofthat of the control or longer are regarded as effective.

The maximum dose level at which they survive for such period as 100 to200 percent of the survival days for the control is signified by ED WithLD 0/ED Index, it is shown that the higher this value is, the less toxicas well as the more effective the sample tested can be.

Results obtained are shown in Table 2.

Table 2 No.* Kind of bleomycin L solED o 32 Index 1 Bleomycin A 94/07812 3 Bleomycin B 75/078 10 7 Bleomycin A l5.6/0.l9 79 93-N,N-Dimethylaminoethylamino- 3.1/0.39 8

' bleomycin l l 3-N,N-Diethylaminopropylamino- 6.6/0.19 35 bleomycin l23-[3-(N-Butyl)-aminopropyl] l 1.3/0.19 59 aminopropylamino-bleomycin 132-(2-Hydoxypropyl)-amino- 8.3/0.39 21 ethylamino-bleomycin 143-(Piperazino)propylamino- 5.6/0.39 14 bleomycin l53[3(N-l-Phenylethyl)- l3.|/0.39 34aminopropyll-aminopropylaminobleomycin 16 2-Aminopropylamino-bleomycin1.9/0.39 5 l7 3-N-Methylaminopropylamino- 2.6/0.19 14 bleomycin l93-(N,N-Dimethyl)-amino- 80/039 20 propylamino-bleomycin 203-(N,N,N-Trimethyl)-amino- 6.7/0.19 35 propyIamino-bleomycin 213-[3-(N,N-Dimethyl)-amino- 10.3/0. l 9 54propyll-aminopropylaminobleomycin 22 3-(N-MethyLN-S-aminopropyl)- 94/01948 aminopropylamino-bleomycin 24 3-(Pyrrolidino)-propylamino 7.5/0.39 l9bleomycin 25 3-(Piperidino)-propylamino 56/039 14 bleomycin 263-(Morpholino)-propylaminol4/0.78 18 bleomycin 28 3-1l-{4-(3-Aminopropyl)}- 8.8/0.1) 46 piperazinol-propylaminobleomycin 293-(3-Pyrrolidino-propyl)- 56/009 62 aminopropylamino-bleomycin 303-(3-Piperidino-propyl)- 66/039 17 aminopropylamino-bleomycin 3|3-(3-Morpholino-propyl)- l4.l/0.l9 74 aminopropylamino-bleomycin 323-(3-Hyrdoxypropyl)- 5.6/0.l9 30 aminopropylamino-bleomycin Table2-Continued Note: The numbers correspond to those mentioned in Table 1of the present specification.

As is clear from above Table 2, bleomycins show high index numberindicating a large difference between a measure amount showing toxicityand that showing lifeprolonged effect as compared with mytomycin C usedas a control medicine. Therefore, it can be said that bleomycin can beused far more safely and is more expectable in the anti-tumor effectthan any other conventionally known anti-tumor substances.

The present invention is illustrated in detail below with reference toexamples. Of course, the present invention shall not be limited to thefollowing examples.

EXAMPLE 1 To a medium having a composition of 6.4 percent of milletjelly, 0.5 percent of glucose, 3.5 percent of soybean powder, 0.75percent of corn steep liquor, 0.3 percent of sodium chloride, 0.1percent of potassium secondary phosphate, 0.05 percent of zinc sulfate,0.01 percent of copper sulfate, 0.2 percent of sodium nitrate and 0.01percent of Toho No. l (tradename for a surface active agent composed ofpolyoxyethylene manufactured by Toho Chemical Industry Co. Ltd., Japan)was added 3-amino-propyl-dimethylsulfonium bromide hydrobromate in aproportion of 0.4 mg/ml to adjust the pH of the medium to 6.5. Each mlof the thus treated medium was separately charged into a Sakaguchi flaskand was then sterilized. Subsequently, Streptomyces verticillus (ATCCNo. 15003) was inoculated in the medium and was cultured at 27C for 8days with stirring at rpm. Thereafter, the culture liquors (4.5 l) werecollected and filtered to obtain 3.0 l of a filtrate (potency 38.8mg/ml, total potency 416.4 mg). This culture filtrate was passed throughand adsorbed on a column packed with 200 ml of Amberlite IRC-SO and waswashed with water and was eluted with 0.5 N hydrochloric acid. 1.0 l ofthe eluate was neutralized, was passed through and adsorbed on a columnpacked with 100 ml of active carbon, was washed and was then eluted byuse of a 1:1 (by volume) mixture of acetone- 0.02 N aqueous hydrochloricacid solution, and fractions active to Mycobacterium 607 were collectedand concentrated to dryness. The resulting residue was dis-

1. BELOMYCINS HAVING A STRUCTURE REPRESENTED BY THE FORMULA,